A. Borrelia burgdorferi, the agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), nitrogen dioxide (NO2), nitrogen trioxide (N2O3) and peroxynitrite (NO3). There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs during tick feeding and as the bacteria migrate through the tick salivary glands during transmission. In FY 2019, we determined the roles of nutrient limitation and reactive nitrogen species (RNS) in survival and gene regulation during the infective cycle. B. burgdorferi must adapt to distinctly different environments in its tick vector and various mammalian hosts. Effective colonization (acquisition phase) of a tick requires the bacteria to adapt to post feeding, tick midgut physiology (nutrient limitation) while successful transmission (transmission phase) to a mammal requires the bacteria to sense and respond to the midgut environmental cues and up-regulate key virulence factors before transmission to a new host (reaction to RNS). Remarkably, these relatively small changes affect two independent regulatory networks that promote acquisition and long-term survival (Hk1-Rrp1) as well as transmission (Rrp2-RpoN-RpoS) of B. burgdorferi. Lysine acetylation serves as a signal and post-translational regulatory response to starvation in the tick midgut during nutrient limitation. Most importantly, recent data from our laboratory shows that dicyclic-GMP, produced by Rrp1, stimulates the phosphatase activity of Hk2. We believe this crosstalk is essential for coordinating these two essential regulatory systems. In related studies, we have shown that RNS that are only present in the midgut of feeding ticks, presents a significant challenge to long-term survival of B. burgdorferi. The damage mediated by RNS stimulates the nucleotide excision repair (NER), base excision repair (BER) and mismatch excision repair (MER) systems which ensures maximum growth and long-term survival. Data from Dr. T. Bourrets laboratory at Creighton University suggests that the response to RNS is mediated by DksA and ppGppp (synthesized by RelA). These data suggest that; (1) dicyclic GMP, triggered by starvation, might be an important regulatory modulator that coordinates Hk1/Rrp1 and Hk2/Rrp2-dependent regulation, and (2) RNS stimulates DksA-dependent gene regulation that is essential for the long-term survival of B. burgdorferi in ticks (2). In a FY 2019 parallel study led by GRC post-doctoral fellow, Dr. S. Bontemps-Gallo, we identified an environmental condition that affects gene expression and long-term survival: nutrient limitation (1). Our study identified nutrient limitation/stationary phase in B. burgdorferi as a critical factor that triggers lysine acetylation. Using a highly sensitive mass spectrometry-based proteomics approach, we characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both mid-log and stationary phases of growth. However, acetylation of target proteins in mid-log phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during stationary phase. To determine the acetyl donor in B. burgdorferi, we used mutants deficient in acetate anabolism. B. burgdorferi strains B31-A3, B31-A3 ackA (acetyl-P- and acetyl-CoA-) and B31-A3 pta (acetyl-P+ and acetyl-CoA-) were grown to stationary phase and the acetylation profiles were analyzed. While only 2 proteins were acetylated in the ackA mutant, 140 proteins were acetylated in the pta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in stationary phase appeared to play a role in decreasing the enzymatic activity of most glycolytic proteins. Currently, we hypothesize that acetylation is used to inactivate enzymes during long-term survival of the bacteria in the tick midgut between blood meals. This strategy would allow the bacteria to activate key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process (1).